Protease inhibitor cocktail b1400

Following the differentiation of C2C12 cells, we added 5 × 10⁸ EVs to each well of a 6-well plate and incubated the cells for 24 h in an EVs-Free medium [35 (link)]. After the incubation period, we collected the cells and re-suspended them in PBS containing a protease inhibitor cocktail (B1400) and phosphatase inhibitor cocktail (B15001) from Bimake (Houston, USA). The cells were placed on ice and sonicated using an amplitude of 30% for 10 s, with this process repeated three times. We collected the cell supernatant via centrifugation at 3000 rpm for 10 min and quantified the total protein in the supernatant using the BCA method. The supernatant was stored at -20 °C for subsequent protein blot analysis.

DON (12, 13-epoxy-3, 4, 15-trihydroxytrichotec-9-en-8-one, C15H20O6, MW: 296.32, purity ≥ 99%, CAS RN: 51481-10-8) was purchased from Sigma-Aldrich (St. Louis, MO, USA). Hematoxylin and eosin staining kit (C0105), enhanced BCA protein assay kit (P0010), RIPA lysate (P0013B), and PMSF (ST505) were purchased from Beyotime Biotechnology (Shanghai, China). Phosphatase inhibitor cocktail (B15001) and protease inhibitor cocktail (B1400) were purchased from Bimake (Houston, TX, USA). Mini-reduced glutathione (GSH) assay kit (A006-2-1,96T), superoxide dismutase (SOD) assay kit (A001-3, WST-1), and catalase (CAT) assay kit (A007-1-1, Ammonium molybdate method) were purchased from Nanjing Jiancheng Institute of Biological Engineering (Nanjing, China). Cell Counting Kit-8 (CCK-8) was purchased from Dojindo (Shanghai, China). The 4% phosphate-buffered paraformaldehyde (C1101) was purchased from Servicebio technology (Wuhan, China). The fluorescent probe dihydroethidium (DHE) was purchased from Beyotime (Haimen, China). The ELISA kit (8-OHdG) was purchased from Elabscience Biotechnology Co. Ltd. (Wuhan, China).